One of the most keenly investigated structural biomaterials that promises great potential in a variety of technological and scientific areas is nacre. Nacre, also known as mother of pearl, is an organic-inorganic composite material produced by certain molluscs such as bivalves, gastropods and cephalopods. The nacre is continually deposited on the inner surface of the shell, protecting the soft tissues from damage by parasites and dirt by entombing these in layers of nacre.
Nacre has long fascinated scientists for its remarkable mechanical properties of strength and toughness attained from inferior performing starting materials. Nacre is made of platelets of aragonite (a polymorph of CaCO3) arranged in, on average, parallel layers. These layers are separated by an organic matrix composed of elastic biopolymers such as chitin, lustrin and silk-like proteins.
The remarkable properties of nacre are a consequence of its detailed nanoscale assembly and construction, as well as ionic crosslinking of the tightly folded biopolymers in the matrix (Tang Z, Kotov N, Maganov S, Ozturk B., “Nanostructured artificial nacre”, Nature Materials, 2003, 2, 413-418).
Nacre is a near ideal platelet composite that is strong in the x (on the platelet face) and y planes (90 degree angle from the face) and is plastic at increasing strain (beyond 0.01). The highly ordered arrangement of crystalline platelets 200-900 nm thick gives the macrostructure its strength. Intercalated matrix proteins, 10-50 nm thick, provide the nacre with elasticity and fracture-stopping capabilities. Fractures are deflected through the matrix which transfers the applied loads at the fracture point.
Generally, synthetic composites cannot be fabricated with the same sophisticated microstructures that enable natural biomaterials such as nacre to perform. Biomineralisation usually occurs via a slow precipitation at close to ambient temperatures and pressures. This process is highly influenced by specialised biomolecules, usually proteins that may absorb on specific crystallographic faces to decrease the growth rate of these faces, giving the crystal a specific morphology. In biomineralisation, the organism can use special compartments of crystallisation to control the temperature, pH and ion concentration of the crystallisation environment.
Many researchers are developing ways of producing nacre-like materials using the substrates and molecular processes involved in nacre shell formation. Nacre formation has been mimicked by exposing specified quantities of key acidic macromolecules and accessory mineral ions such as magnesium ions to chitosan templates to modulate calcium carbonate crystal morphology, size and polymorph.
Replicating the slow process of biomineralisation in vitro is difficult, but several methods of mineralising matrices have been developed, to generate nacre-like composite materials.
In the ammonium carbonate diffusion method, ammonium carbonate vapour is diffused over organic surfaces leading to rapid calcium carbonate precipitation (Tawashi R, Bisaillon S, Wolter K. In vitro formation of calcite concretions. Experientia, 1974, 30(10) 1153-1154; Addadi L, Weiner S. Interaction between acidic proteins and crystals: Stereochemical requirements in biomineralization. Proceedings of the National Academy of Science USA, 1985, 82, 4110-4114).
Another method is double diffusion, where solutions containing the respective cation and anion of the target mineral are separated by a polymer or hydrogel template. The diffusion through the template induces mineralisation within the template. However, the formation of crystals within the template can lead to limited growth at the periphery due to decreased diffusion of the ions through the material.
In US patent application US 2007/0225328, Fritz et al. describe using double diffusion of calcium and carbonate ions to mineralise a template comprising the water-insoluble matrix of mussel, snail or other chitin-containing protecting armament of a sea water animal. The double diffusion method involves counter-diffusion of two solutions across an insoluble matrix at opposite sides. A decrease in pH affects the surface properties of the matrix, encouraging calcium carbonate nucleation and growth. The template is prepared for mineralisation by cleaning and crushing the chitin-containing shell, incubating with sodium hypochlorite to remove organic contaminants, then removing the existing calcium carbonate using a 30 day dialysis against EDTA with sodium azide.
While the process is capable of providing a synthetic nacre-like product, it is expensive and time consuming, because it starts with a natural chitin template that must first be stripped of its natural mineralisation only to be replaced with synthetic mineralisation.
Most methods used seek to mineralise an artificially made organic template.
The alternative soaking method was developed in the late 1990s in Akashi's group (Taguchi, T, Kishida, A and Akashi, M. Chem. Lett., 1998, 8, 711-712). In the alternate soaking method, a polymer template such as a chitosan film is repeatedly alternately soaked with solutions containing the respective cation and anion of the target material.
In the Kitano method, effusion of CO2 gas from saturated CO2-calcium carbonate solution increases the pH of the crystallisation solution leading to crystal nucleation and growth (Kitano, Y; Bull. Chem. Soc. Jpn., 1962, 35(12), 1973-1980). However, the Kitano method tends to produce mineralisation at the peripheral surface of the polymer template only, so minimal penetration into the polymer matrix is achieved. Furthermore, the extent of mineralisation is limited due to the use of a saturated calcium carbonate solution.
The Kitano and alternate soaking methods can be combined by alternate soaking of the chitosan films with precursor calcium and carbonate solutions followed by treatment with saturated mineral solution (Munro, N H; Green, D W; Dangerfield, A; and McGrath, K M; Dalton Trans., 2011, 40, 9259).
In the above methods, the template to be mineralised generally takes the form of a thin film. Although differently shaped templates can provide a range of differently shaped composite materials, the thickness of these materials is generally limited. Using a thicker template reduces mineralisation in the internal surfaces, leading to a weaker product. Therefore, while good mineralisation of an organic film can sometimes be achieved, growth is effectively limited to two dimensions. However, some attempts at making a genuinely three-dimensional material have been made.
Tang et al. formed an organic/inorganic composite material by depositing preformed inorganic crystals on an appropriately charged organic template (Tang, Z.; Kotov, N. A.; Magonov, S.; Ozturk, B., Nanostructured artificial nacre. Nat. Mater. 2003, 2(6), 413-418.). Growth in the third dimension (perpendicular to the largest surface) was achieved by depositing another organic layer on the top, then another layer of inorganic crystals i.e. “layer-by-layer” deposition.
Kato has also made advances in this area by forming the inorganic component in situ. Kato deposited a thin film of chitosan or chitin onto a glass substrate and grew calcium carbonate (the inorganic component) on top (Kato, T.; Suzuki, T.; Irie, T., Layered thin-film composite consisting of polymers and calcium carbonate: A novel organic/inorganic material with an organized structure. Chem. Lett. 2000, 2, 186-187). A layer of calcium carbonate of about 0.8 μm was achieved, using saturated calcium carbonate solution i.e. the “Kitano” method. Additional growth in the third dimension was achievable by deposition of another organic layer on top followed by more calcium carbonate deposition. The calcium carbonate was present as calcite.
While a large range of composite materials have been formed by mineralisation of a synthetic porous template, to date none of these materials demonstrate properties close to those seen in nacre. Mineralisation is generally limited to the periphery of the template with minimal mineralisation in the internal surfaces of the organic matrix. In addition, mineralisation tends to occur in two dimensions only. Where growth in the third dimension is achieved, it is via a laminar product built of alternate organic and inorganic layers. These layers are held together by weak intermolecular interactions, hence the material lacks the desirable structural factors of natural nacre.
Accordingly, it is an object of the invention to provide a method for producing mineralised composite materials, that goes at least some way towards overcoming the disadvantages of known methods, or to provide the public with a useful choice.